In the identification and structural analysis of macromolecular compounds such as sugar chains and peptides, ion trap mass analysis devices equipped with a MALDI (matrix-assisted laser desorption/ionization) ion source and a three-dimensional quadrupole ion trap are widely used. The technique of mass analysis of various ions held temporarily in the ion trap includes cases where the mass separation function of the ion trap itself is used and cases where ions are transferred all at once from the ion trap and detected after performing mass separation of the ions by means of a time-of-flight mass spectrometer provided outside the ion trap, but here, these will be referred to together as ion trap mass analysis devices.
The general analysis technique for macromolecular compounds using an ion trap mass analysis device is as follows.
The target compound which is the object of analysis is ionized by the MALDI process and captured in the ion trap, after which an ion selection operation is performed, whereby ions derived from the target compound and having a specified mass/charge ratio m/z are selectively left behind in the ion trap as precursor ions, while other unneeded ions are eliminated out of the ion trap. Subsequently, a collision-induced dissociation (CID) gas is introduced into the ion trap, and the precursor ions are excited by the action of a high frequency electric field and made to collide with the CID gas, thereby promoting the dissociation of the precursor ions. In some cases, the target structure will not be adequately dissociated through a single CID operation, so the selection of precursor ions and the CID operation may be repeated multiple times. Product ions which have been finely fragmented by performing one or more CID operations on the ions derived from the target compound are then subjected to ion detection involving mass scanning, to acquire an MSn spectrum, and this MSn spectrum is then used, for example, for a database search or the like to identify the compound or infer its structure.
To increase the precision of compound identification and structural analysis as described above, or to shorten the time requirement for measurement and analysis and increase the throughput, it is important to select ions with an appropriate mass/charge ratio as the precursor ions which will be the object of the CID operation. As the general conventional precursor ion selection method, a technique has been employed wherein the peaks appearing in a mass spectrum (MS1 spectrum) acquired by performing regular mass analysis without performing a CID operation are detected, all the peaks that satisfy a predetermined criterion, such as having a signal intensity above a threshold value, are extracted, and the ions corresponding to those peaks are selected as the precursor ions. Normally, numerous peaks which satisfy the predetermined criterion will be present on a mass spectrum, so if MS2 analysis is to be executed on all of them, the measurement alone will take a very long time. Furthermore, as the number of measurements increases, the quantity of sample consumed also becomes accordingly greater, and thus a large amount of sample needs to be prepared.
To avoid the problems described above, a method is conceivable whereby, rather than extracting all the peaks on the mass spectrum which satisfy predetermined criteria, a restriction is imposed wherein, for example, a priority order is applied based on order of intensity, and only a predetermined number of peaks are extracted. However, it is not necessarily the case that peaks of relatively high signal intensity will characterize the structure of the target compound, so simply extracting the peaks mechanically in intensity order or the like may not allow one to select effective precursor ions for identification and structural analysis.
A concrete example will be taken and described in detail. Biological macromolecular compounds such as peptides, which are often taken as the object of analysis in an ion trap mass analysis device, often contain easily detachable modifiers, functional groups and the like. Sialic acid, sulfate groups, phosphate groups and the like are well known as typical modifiers. For example, it is known that sialic acid is preferentially detached through low energy CID in the case of sugar chains to which sialic acid is bonded, which are a type of acidic sugar, or glycopeptides to which a sialic acid-bonded sugar chain has been attached, but this sort of detachment of sialic acid is easily produced not just by the CID process but also by in-source decay and collision with cooling gas in the ion trap (for example, see Patent Literatures 1 and 2). Thus, when the sample contains a compound to which multiple easily detachable modifiers as described above are bonded, in the mass spectrum obtained without executing CID, there will appear a mixture of peaks derived from ions for which the modifiers have not been at all detached from the basic structure, peaks derived from ions for which a portion of modifiers have been detached from the basic structure, and peaks derived from ions for which all the modifiers have been detached from the basic structure. Thus, the mass spectrum becomes quite complex. Furthermore, since the intensity of ions containing the basic structure is distributed over multiple peaks, the intensity of each peak may become relatively lower. In such cases, with the technique of extracting a number of peaks determined according to intensity order, as described above, there is the concern that ions containing the basic structure which provide a good representation of the characteristics of the compound will not be selected as precursor ions.